JOURNAL OF VIROLOGY, Sept. 2005, p. 12100–12105 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.18.12100–12105.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 18
Unique Acquisition of Cytotoxic T-Lymphocyte Escape Mutants in Infant Human Immunodeficiency Virus Type 1 Infection† Thillagavathie Pillay,1 Hua-Tang Zhang,1 Jan W. Drijfhout,2 Nicola Robinson,1 Helen Brown,1 Munira Khan,3 Jagadesa Moodley,3 Miriam Adhikari,4 Katja Pfafferott,1 Margaret E. Feeney,5 Anne St. John,6 Edward C. Holmes,7 Hoosen M. Coovadia,8 Paul Klenerman,1 Philip J. R. Goulder,1 and Rodney E. Phillips1* The Peter Medawar Building for Pathogen Research and Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom1; Department of Immunohaematology, Leiden University Medical Centre, Leiden, The Netherlands2; Nelson R. Mandela Medical School, Department of Obstetrics and Gynaecology, University of Natal, Durban, South Africa3; Nelson R. Mandela Medical School, Department of Paediatrics and Infant Health, University of Natal, Durban, South Africa4; Partners AIDS Research Center and Infectious Disease Division, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts5; Queen Elizabeth Hospital, Bridgetown, Barbados6; Department of Evolutionary Biology, University of Oxford, Oxford, United Kingdom7; and Centre for HIV and AIDS Networking, University of Natal, Durban, South Africa8 Received 27 January 2005/Accepted 24 May 2005
The role of cytotoxic T-lymphocyte (CTL) escape in rapidly progressive infant human immunodeficiency virus type 1 (HIV-1) infection is undefined. The data presented here demonstrate that infant HIV-1-specific CTL can select for viral escape variants very early in life. These variants, furthermore, may be selected specifically in the infant, despite the same CTL specificity being present in the mother. Additionally, pediatric CTL activity may be compromised both by the transmission of maternal escape variants and by mother-to-child transmission of escape variants that originally arose in the father. The unique acquisition of these CTL escape forms may help to explain the severe nature of some pediatric HIV infections. In developing countries, one-third of human immunodeficiency virus type 1 (HIV-1)-infected children have rapidly progressive disease and die in infancy (23). It is unknown why infants have particularly poor control of HIV-1 (5, 6, 8, 9). Cytotoxic T-lymphocyte (CTL) responses can control adult HIV-1 and simian immunodeficiency virus infections (12, 18, 20) but select for CTL escape mutations with a subsequent loss of immune control (2, 11, 14, 19). Few studies have described CTL in early perinatal HIV-1 (3, 17). However, it is unclear whether rapid progression in infants occurs in association with an undetectable HIV-specific CTL response or with an ineffective HIV-specific CTL response (21). This distinction is of relevance to pediatric HIV vaccine design strategies. Here we provide evidence that very early in the first year of life, CTL drive the selection of de novo escape variants, which together with mother-to-child transmission of viruses preadapted to the HLA class I alleles expressed in the infant, are likely to contribute to a lack of immune control in pediatric infection. We studied five rapidly progressing infants and their mothers from Durban, South Africa, from pregnancy to up to 1.5 years after birth (see Table S1 in the supplemental material). Three of the five infants died with HIV-1 disease within 3 to 23 months. A fourth infant commenced antiretroviral therapy at 1 year of age after the onset of AIDS. The fifth infant was * Corresponding author. Mailing address: University of Oxford, The Peter Medawar Building for Pathogen Research, OX1 3SY Oxford, United Kingdom. Phone: 44-1865-281880. Fax: 44-1865-281890. Email:
[email protected]. † Supplemental material for this article may be found at http://jvi .asm.org/.
withdrawn from the study by his guardians following the AIDSrelated death of his mother. To assess infant immunity-driven viral evolution, we examined HIV-1 viral genes encoding the most immunogenic viral proteins (1), Gag and Nef. gag and nef were sequenced from plasma RNAs collected from infants and mothers from pregnancy onwards. Phylogenetic analysis (22) confirmed the relatedness of clones from mother-child pairs, all of which clustered with clade C viruses (see Fig. S1 in the supplemental material). At six epitopes from three infants, we identified de novo CTL escape (Table 1; see Table S2 in the supplemental material). Using the method of CODEML selection analysis as previously described (7), four of these antigenic sites were shown to be under positive selection in these infants (Table 1) (dn/ds ⬎ 1; P ⬍ 0.05). Maximum likelihood phylogenetic analysis indicated that in each case, the CTL escape variant had evolved subsequently in the infant (Fig. 1 and data not shown). No variation was observed at these sites in ⬎50 clones from each mother sampled across different time points. This observation, together with phylogenetic evidence, suggests that the CTL escape viruses had arisen in the infants. We next examined epitopes presented by alleles shared by mother and child to determine whether escape variants could be specifically selected in rapidly progressing infants. An analysis of nef from infant I4 demonstrated the selection of a Pro-to-Ser or -Gln change at position 2 of the HLA-B*4201 epitope, TPGPGVRYPL, between 10 and 26 weeks postpartum (Table 1; Fig. 2a). These variant peptides generated specifically in the infant were not recognized (Fig. 2b) and did not bind to HLA-B*4201 (Fig. 2c). These CTL escape mutants
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Infant or mother
Mother M1 Infant I1 Mother M2 Infant I2 Mother M3 Infant I3
Mother M4 Infant I4 Mother M5 Infant I5 Mother M5 Infant I5 Mother M5 Infant I5
HLA type
A*2301/6802, B*0801/ 1510, Cw*0301/0801 A29/6802, B*4403/ 1510, Cw*0701/0801 Not done A*2601/68, B*0702/⫺, Cw*0702/⫺ A*0301/7401, B*1510/ 3501, Cw*0401/⫺ A*0301/68, B*0702/ 1510, Cw*0401/0702
A*3001/30, B*1503/ 4201, Cw*0202/1701 A*0301/3001, B*4201/ 5802, Cw*0602/1701 A*3001/66, B*4201/ 5802, Cw*0602/1701 A*68/66, B*57/5802, Cw*0602/0701
CTL epitope and HLA restriction
Nef73–82, A*03 Nef73–82, A*03
Nef128–137, B*42 Nef128–137, B*42 Nef116–124, B*57 Nef116–124, B*57 Nef82–90, B*57
Gag240–249, B*57
Gag147–155, B*57
0.00 0.09 0.91
0.00 1.00
0.10 0.90
1.00 0.61 0.05
0.91 0.09 0.22 0.03 0.03 0.20
Frequency of variant in infant
33 33 33
33 33
33 33
33 33
10 26 26
2 2 42 80 80 80
Age (wks) at which variant was first detected
830 0
160 280
1,150 150
1,151 0 0
1,550 350 520 0 0 1,250
ELISPOT response for infant (no. of spots/million peripheral blood mononuclear cells)
280 0 180
0.00 0.78 0.20 0.02
TABLE 1. De novo CTL escape in infant HIV-1 Epitope sequencea
OVPLRPMTYK ........F. ........F. ....K...F. .......NY. ...V....F. .......SYR .......SY. TPGPGVRYPL .......... .......... .S........ .Q........ HTOGYFPDW ....F.... ....F.... N...F.... KAAFDLSFF ......G.. ......G.. .G....G.. TSTLQEQIAW ........T. ........T. ........A. ..N.....A. ISPRTLNAW ......... ......... M........ L........ PL........
HIV-1 subtype C consensus sequences are indicated in bold. CODEML selection analysis was performed for amino acids under positive selection. For Nef epitope QVPLRPMTYK, the values represent those for amino acid position 81. NS, not significant.
Mother M5 Infant I5
a b
P valueb
⬍0.001
0.003
NS
NS
0.006
0.006
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driven by selection pressure in the infant were absent from the B*4201-positive mother, in spite of the presence of the same TL10-specific CTL response in the mother (Fig. 2d). The combination of a consistent high-frequency TL10-specific response and a persistent high maternal viral load of ⬎100,000 HIV RNA copies/ml of plasma suggests that this particular response was ineffective (4, 24), whereas the same response in the infant “drove” the evolution of the escape variants early on in the infection. These data demonstrate that, at least for certain epitopes, selection for escape may operate during early pediatric HIV infection but may be absent from chronic adult infections. It has previously been shown that children may be in a particularly disadvantaged position with respect to the epitopes available for an effective HIV-specific CTL response as a result of mother-to-child transmission of a virus that has adapted successfully to the maternal class I alleles (10, 15). Since infants typically share 50% of their HLA class I alleles with their mothers, it might be predicted that the remaining 50% of the infant’s HLA alleles that are paternally inherited could be utilized more successfully to mount responses against maternally transmitted virus. However, in some cases, the maternal HIV infection may result from the transmission of virus from the child’s father. To examine the question of whether viruses adapted to paternal HLA alleles may in this way be indirectly transmitted to the child via the mother, we sought “footprints” of paternal HLA alleles in viruses transmitted from mother to child. Previous studies of gag sequences from HLA-B*57-positive subjects have identified “footprints” of HLA-B*57 which persist following transmission to B57-negative subjects (16). In particular, these arise at Gag residues 219 (H219Q) and 248. For clade B infections, the characteristic B*57 footprint is G248A, whereas for clade C infections, the B57 footprint at this site is A248T (P ⫽ 0.036) (16). The occurrence of such HLA-B*57-associated polymorphisms in the HLA-B*57-negative mother M5 (Fig. 3a) is thus indicative of the transmission of virus from an HLA-B*57-positive subject to the mother (P ⫽ 0.008) (16). Since child I5 had HLA-B*57, one may speculate that the HLA-B*57-positive father in this instance directly transmitted a virus carrying these HLA-B*57-associated mutations to the HLA-B*57-negative mother, M5. These B*57 “footprints” persisting in the mother were, in turn, transmitted to the HLA-B*57-positive child. In this example, the transmitted variant A248T did not affect binding to HLA-B*57, and thus a TW10 response (and further immune pressure on the virus) could be generated by the child. The transmission of an escape variant unable to bind to the HLA molecule would have precluded a response in the infant. One example of mother-
FIG. 1. Selection of intraepitope variants in infants. Maximum likelihood phylogenetic trees of maternal and infant sequences of nef and gag from two mother-infant pairs are shown. Amino acid position T110 of the maternal Gag240–249 epitope, TSTLQEQITW (in black), and its variants T242N (in red) and T248A (in blue) are mapped on tree a.
Amino acid position H116 of epitope Nef116–124, HTQGFFPDW (in black), and its variant H116N (in red) are mapped on tree b for mother-infant pair 5. This infant was PCR negative for HIV-1 at birth and positive at 12 weeks of age. Infant sequences at 33 weeks of age are shown. Amino acid position P129 of epitope Nef128–137, TPGPGVRYPL, and its variants P129S (in red) and P129Q (in green) are mapped onto phylogenetic tree c for mother-infant pair 4. In all trees, maternal and infant sequences are depicted; infant sequences are shown in gray boxes. In all instances, viruses bearing the maternal amino acid were transmitted to the infant. The mutations H116N, T242N, P129S, and P129Q evolved subsequently.
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FIG. 2. Early escape from infant CTL recognition. (a) Amino acid sequences of HIV-1 Nef in infant I4 and his mother M4 showing HLA B*4201-restricted Nef128–137 epitope TPGPGVRYPL. The wild-type virus was transmitted from mother M4. The times depicted are relative to birth. (b) In ex vivo enzyme-linked immunospot (ELISPOT) assays using infant peripheral blood mononuclear cells sampled at 10 weeks, CTL from I4 recognized the wild-type peptide. By 26 weeks, Nef variants TSGPGVRYPL and TQGPGVRYPL emerged; these evaded CTL recognition. This infant shared HLA B*4201 with his mother. (c) Binding of TPGPGVRYPL and its variants, TSGPGVRYPL and TQGPGVRYPL, to HLA B*4201 molecules in a competitive fluorescent peptide inhibition assay (13). The sigmoid dose-response curve shows a reduction in the percent inhibition of a fluorescent peptide (y axis) as the concentration of test peptide (x axis) is decreased. BCL expressing HLA B*4201 were subjected to a mild acid treatment at pH 3.1 and incubated with a fluorescent peptide [APAPAPC(fl)WPL] and the test peptides TPGPGVRYPL, TSGPGVRYPL, and TQGPGVRYPL. An unlabeled high-affinity binding peptide (SPSVDKARAEL) was included as a positive control. The variant peptides TSGPGVRYPL and TQGPGVRYPL failed to competitively displace 50% of the fluorescently labeled peptide, while TPGPGVRYPL competitively inhibited the fluorescently labeled peptide in a dose-dependent fashion as effectively as the positive control. This indicated that relative to TPGPGVRYPL, the variants TSGPGVRYPL and TQGPGVRYPL bound poorly to HLA B*4201. (d) Maternal CTL recognize TPGPGVRYPL through pregnancy and after birth in mother M4. In ex vivo ELISPOT assays (done in duplicate), maternal peripheral blood mononuclear cells consistently recognized the HLA B*4201-restricted epitope. Despite this, variation at this site was not detectable in mother M4. Times depicted on the graph are relative to birth (time zero).
to-child transmission of such a variant has been described for the B27-KK10 epitope, where Arg is required for binding to B27 (10, 11). We identified another example of this in a Barbadian mother-child pair (Fig. 3b), but with HLA-B27 being
shared by the mother and child. In this instance, the presence of the B27 footprint R264X in the virus of the B27-negative father and a sequence analysis demonstrating the shared phylogeny of the paternal and maternal viruses (Fig. 3c) indicated
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FIG. 3. Maternal and infant viruses contain footprints of paternal alleles. (a) Infant 5 inherited HLA B57 paternally. “Footprints” of HLA-B57, namely, A248T and H219Q (16), were detected in the maternal virus. The H219Q variant was transmitted to the HLA-B57-positive child. At position A242 of the epitope TSTLQEQIAW, the infant generated an HLA B57-restricted CTL escape form, A242N. (b) The transmission of a virus containing an HLA B*27 CTL escape sequence from an HLA B*27-positive mother to her HLA B*27-positive child was detected in a Barbadian family, Barb-1. Although the father did not have the HLA B*27 allele, the CTL escape form was detected. (c) Maximum likelihood phylogenetic tree showing the genetic relationships of HIV-1 subtype B viruses for the Gag proteins p17 and p24. Families from Boston (043TCH and 068TCH) and Barbados (Barb-1 and Barb-2) (M, mother; D, father; C, child) are indicated by red branches, which are all supported by bootstrap values of 100%. The Barbadian family detailed in panel b is highlighted in the gray box. Reference HIV-1 subtype C and B sequences were obtained from the Los Alamos Database. Close phylogenetic relationships between mother, father, and child sequences were observed in each case.
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that in this case, transmission of the virus occurred from mother to father. Analyses of these and other families show that HIV infection in one parent not infrequently is the result of transmission by the other parent (Fig. 3c), and thus the acquisition of a virus adapted to paternal and maternal HLA alleles may occur in the infant via mother-to-child transmission. These data show that there are several unique influences that may compromise the effectiveness of the early pediatric HIV-specific immune response that involve CTL escape. These include the transmission of escape variants generated in the mother and also those originally generated in the father and the early development of escape mutations in epitopes at which selection for escape does not necessarily occur in adult infections. The occurrence of de novo escape during early pediatric infections implies, on the one hand, a suboptimal immune response akin to the development of drug-resistant mutations in the presence of suboptimal antiretroviral therapy. On the other hand, the presence of functional CTL generated against HIV in early infancy suggests the possibility that early immunomodulatory interventions may have promise to improve the efficacy of these CTL responses and bring about more successful HIV-specific control of pediatric infections in the future. REFERENCES 1. Addo, M. M., X. G. Yu, A. Rathod, D. Cohen, R. L. Eldridge, D. Strick, M. N. Johnston, C. Corcoran, A. G. Wurcel, C. A. Fitzpatrick, M. E. Feeney, W. R. Rodriguez, N. Basgoz, R. Draenert, D. R. Stone, C. Brander, P. J. Goulder, E. S. Rosenberg, M. Altfeld, and B. D. Walker. 2003. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J. Virol. 77:2081– 2092. 2. Barouch, D. H., J. Kunstman, M. J. Kuroda, J. E. Schmitz, S. Santra, F. W. Peyerl, G. R. Krivulka, K. Beaudry, M. A. Lifton, D. A. Gorgone, D. C. Montefiori, M. G. Lewis, S. M. Wolinsky, and N. L. Letvin. 2002. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 415:335–339. 3. Brander, C., P. J. Goulder, K. Luzuriaga, O. O. Yang, K. E. Hartman, N. G. Jones, B. D. Walker, and S. A. Kalams. 1999. Persistent HIV-1-specific CTL clonal expansion despite high viral burden post in utero HIV-1 infection. J. Immunol. 162:4796–4800. 4. Buseyne, F., S. Blanche, D. Schmitt, C. Griscelli, and Y. Riviere. 1993. Detection of HIV-specific cell-mediated cytotoxicity in the peripheral blood from infected children. J. Immunol. 150:3569–3581. 5. Buseyne, F., M. Burgard, J. P. Teglas, E. Bui, C. Rouzioux, M. J. Mayaux, S. Blanche, and Y. Riviere. 1998. Early HIV-specific cytotoxic T lymphocytes and disease progression in children born to HIV-infected mothers. AIDS Res. Hum. Retrovir. 14:1435–1444. 6. Cheynier, R., P. Langlade-Demoyen, M. R. Marescot, S. Blanche, G. Blondin, S. Wain-Hobson, C. Griscelli, E. Vilmer, and F. Plata. 1992. Cytotoxic T lymphocyte responses in the peripheral blood of children born to human immunodeficiency virus-1-infected mothers. Eur. J. Immunol. 22: 2211–2217. 7. Draenert, R., S. Le Gall, K. J. Pfafferott, A. J. Leslie, P. Chetty, C. Brander, E. C. Holmes, S. C. Chang, M. E. Feeney, M. M. Addo, L. Ruiz, D. Ramduth, P. Jeena, M. Altfeld, S. Thomas, Y. Tang, C. L. Verrill, C. Dixon, J. G. Prado, P. Kiepiela, J. Martinez-Picado, B. D. Walker, and P. J. Goulder. 2004. Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J. Exp. Med. 199:905–915. 8. Feeney, M. E., Y. Tang, K. A. Roosevelt, A. J. Leslie, K. McIntosh, N.
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